RESEARCH ARTICLE

Endurance exercise protects aging Drosophila from high-salt diet(HSD)-induced climbing capacity decline and lifespan decreaseby enhancing antioxidant capacityDeng-Tai Wen1,*, Wei-Qing Wang1, Wen-Qi Hou1, Shu-Xian Cai2 and Shuai-Shuai Zhai1

ABSTRACTA high-salt diet (HSD) is a major cause of many chronic and age-related defects such as myocardial hypertrophy, locomotorimpairment and mortality. Exercise training can efficiently preventand treat many chronic and age-related diseases. However, itremains unclear whether endurance exercise can resist HSD-induced impairment of climbing capacity and longevity in agingindividuals. In our study, flies were given exercise training and fed aHSD from 1-week old to 5-weeks old. Overexpression or knockdownof salt and dFOXO were built by UAS/Gal4 system. The resultsshowed that a HSD, salt gene overexpression and dFOXOknockdown significantly reduced climbing endurance, climbing index,survival, dFOXO expression and SOD activity level, and increasedmalondialdehyde level in aging flies. Inversely, in a HSD aging flies,endurance exercise and dFOXO overexpression significantlyincreased their climbing ability, lifespan and antioxidant capacity, butthey did not significantly change the salt gene expression. Overall,current results indicated that a HSD accelerated the age-relateddecline of climbing capacity and mortality via upregulating saltexpression and inhibiting the dFOXO/SOD pathway. IncreaseddFOXO/SOD pathway activity played a key role in mediatingendurance exercise resistance to the low salt tolerance-inducedimpairment of climbing capacity and longevity in aging Drosophila.

This article has an associated First Person interview with the firstauthor of the paper.

KEY WORDS: Aging, Exercise, High-salt diet, Climbing ability,Lifespan, dFOXO/SOD

INTRODUCTIONSodium chloride from dietary salt supplies essential electrolytes tothe human body, and it plays a vital role in maintaining the stabilityof the intracellular and extracellular environments (Li et al., 2018).Despite its essential involvement in many physiological activities,too much salt uptake has been identified as detrimental for many

well-recognized and age-related diseases such as myocardialhypertrophy, hypertension and cancer (Maruyama et al., 2015;Xu et al., 2018a,b). For example, a high-salt diet (HSD)-inducedhypertrophy is a very important and modifiable risk factor forcardiovascular disease and mortality in aging individuals(McGuffin, 2003; Samadian et al., 2016). High-salt intake maypredispose young individuals to developing diseases later. Althougha modest reduction in people’s salt intake worldwide would result ina major improvement in public health, unfortunately, large numbersof young, adult and elderly individuals are still exposed to high-saltfoods because they are accustomed to the taste (Liem, 2017).Therefore, it is important for public health to find other ways toprevent diseases and mortality caused by HSDs.

Exercise training is an efficient strategy for the prevention andtreatment of many chronic diseases caused by diet or aging such asmyocardial hypertrophy, hypertension and obesity (Pedersen andSaltin, 2015). For example, hypertension is a major risk for heartdisease, stroke, kidney disease and other complications, includingdementia. Physical exercise, which promotes hemodynamic andhumoral changes in healthy subjects, may positively affecthypertensive subjects (Burgess et al., 1996). A HSD is a major causeof hypertension, and it accelerates the secondary aging process and canlead to premature death (Samadian et al., 2016). Long-term exerciseresults in a number of physiological adaptations such as an increaseangiogenesis and antioxidant ability that improves muscle performanceand enhances the resistance of the muscle to fatigue (Ahmadiasl et al.,2012; Petersen andGreene, 2008). However, a HSD can impair skeletalmuscle performance by inhibiting its angiogenesis and increasingoxidative stress (Bernardi et al., 2012; Lenda and Boegehold, 2002; Liet al., 2018; Petersen and Greene, 2008). Therefore, these studiessuggest that in regards to health, exercise training and a HSD arein opposition to each other. However, it remains unclear whateffect exercise and a HSD have on chronic diseases, exercise ability,aging and mortality when they are both present over a lifetime.

The fruit fly Drosophila melanogaster provides significantpractical advantages over other model systems to study themolecular mechanisms of exercise training, nutrition and aging,which include developed exercise and diet programs, a shortlifespan (2–3 months), low genetic redundancy compared withmammals, and a plethora of tools available to easily manipulategene expression (Mullinax et al., 2015; Rose, 1999; Sujkowski andWessells, 2018). For instance, in flies the organization andmetabolism of skeletal muscle fibers is similar to that inmammals. After a training program on Power Tower or TreadWheel, trained flies experience a suite of adaptations, and theirclimbing speed, endurance, flight performance, mitochondrialactivity, lipolysis and lifespan are all increased (Lowman et al.,2018; Tinkerhess et al., 2012). However, during the short lifespan offruit flies, defects in flight, climbing and locomotion becomeReceived 31 May 2019; Accepted 20 April 2020

1Department of Physical Education, Ludong University, City Yantai 264025, ShanDong Province, China. 2Co-Innovation Center for Utilization of Botanical FunctionalIngredients, Department of Agronomy, Hunan Agricultural University, Changsha,410128, China.

*Author for correspondence (dt.wen@foxmail.com)

D.-T.W., 0000-0001-7188-1149; W.-Q.W., 0000-0002-5760-0555; W.-Q.H.,0000-0002-3160-4670

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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progressively evident (He and Jasper, 2014). In addition, in a studyof exercise and gene function, it has been reported that theDrosophila homolog of the vertebrate exercise response gene PGC-1α is necessary to induce exercise-dependent phenotypes. Reductionof PGC-1α expression acutely reduces negative geotaxis ability andexercise-induced improvement in both negative geotaxis and time toexhaustion. On the contrary, muscle/heart specific PGC-1αoverexpression improves negative geotaxis and cardiac performancein untrained flies (Tinkerhess et al., 2012). Moreover, a diet withcalorie restriction can extend longevity of flies, but a high-fat diet caninduce obesity and aging phenotype, and acutely reduce lifespan offlies (Birse et al., 2010; Guida et al., 2019; Liu et al., 2012).Endurance exercise protects flies from obesity and lifespan reductioninduced by a high-fat diet when exercise and a high-fat diet arepresent at the same time (Wen et al., 2018). Therefore, because ofthese characteristics, Drosophila is emerging as a useful modelorganism to study molecular pathways of exercise, nutrition andaging in concert with mammalian models.It has been reported that a strict HSD acutely decreases the

lifespan of flies, and the salt gene is an important gene thatcontributes to salt tolerance (Stergiopoulos et al., 2009). However, itremains unknown whether endurance exercise can combat theadverse effects of a HSD on climbing ability and lifespan. In thisstudy, wild-type flies took part in endurance exercise and fed a HSDfrom 1 week to 5 weeks of age to explore the effect of exercisecombined with a HSD on climbing ability (climbing speed andclimbing endurance), lifespan, salt tolerance and antioxidant

capacity. Next, differential expression of salt and dFOXO werebuilt by UAS/arm-gal4 system to explore the potential molecularmechanisms of exercise resistance to a HSD.

RESULTS AND DISCUSSIONA HSD accelerated the decline of climbing capacity andmortality in aging DrosophilaIn mammals, it has been reported that a HSD inhibits angiogenesis inresponse to chronic muscle stimulation and impairs skeletal muscleperformance (Petersen and Greene, 2008). A HSD intake acutelyimpairs muscular exercise ability due to the calcium deficit in musclecells via the destruction of sodium-calcium exchange homeostasis(Frisbee et al., 2000). In flies, their muscles are used for flying,crawling and climbing. Therefore, the function and contractilephysiologicalmechanism of skeletalmuscle inDrosophila are similarto those in mammals (Piccirillo et al., 2014). However, it remainsunclear whether a HSD can affect climbing capacity.

The results showed that in 1-week-old flies, a 2%-salt diet (2%-SD), a 4%-SD and an 8%-SD significantly reduced the climbingfatigue time (CFT) (P<0.05, P<0.01, P<0.001, respectively)(Fig. 1B). In 3-week-old flies, a 2%-SD, a 4%-SD and an 8%-SDalso significantly reduced the CFT (P<0.001, P<0.001, P<0.001,respectively) (Fig. 1C). In 5-week-old flies, a 2%-SD, a 4%-SD andan 8%-SD significantly reduced the CFT (P<0.01, P<0.001,P<0.001, respectively) (Fig. 1D). In addition, the resultsdisplayed that in 0%-SD flies, 2%-SD flies, 4%-SD flies and 8%-SD flies, senility significantly reduced the CFT (P<0.01, P<0.01,

Fig. 1. The effect of HSDs on climbing capacity and mortality in aging flies. (A) An image of HSD flies drinking water. (B) Time to fatigue of 1-week-oldflies. (C) Time to fatigue of 3-week-old flies. (D) Time to fatigue of 5-week-old flies. (E) Time to fatigue in w1118 flies. (F) Time to fatigue in 2%-SD flies.(G) Time to fatigue in 4%-SD flies. (H) Time to fatigue in 8%-SD flies. (I) The climbing index changes with aging. (J) The climbing index in HSD flies. (K) Thecurves of survival and the average lifespan. Using a non-parametric followed by a log-rank test to analyze ‘survival’ and ‘time to fatigue’. The one-wayanalysis of variance (ANOVA) with least significant difference (LSD) tests were used to identify differences among the ‘w1118’, ‘w1118+2%salt’, ‘w1118+4%salt’ and ‘w1118+8%salt’ flies. Data are represented as means±s.e.m. *P<0.05; **P<0.01; ***P<0.001.

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P<0.01, P<0.05, respectively) (Fig. 1E–H). Next, the resultsdisplayed that in 0%-SD flies, 2%-SD flies, 4%-SD flies and 8%-SD flies, senility significantly reduced the climbing index (CI)(P<0.01, P<0.01, P<0.01, respectively) (Fig. 1I). In 1-week-oldflies, a 4%-SD and an 8%-SD significantly reduced the CI (P<0.05,P<0.01, respectively), but a 2%-SD had no remarkable influence onCI (P>0.05) (Fig. 1J). In 3-week-old flies, a 2%-SD, a 4%-SD andan 8%-SD significantly reduced the CI (P<0.05, P<0.01, P<0.01,respectively) (Fig. 1J). In 5-week-old flies, a 2%-SD, a 4%-SD andan 8%-SD significantly reduced the CI (P<0.05, P<0.01, P<0.01,respectively) (Fig. 1J). These results suggested that a HSD couldaccelerate the age-related locomotor impairment in agingDrosophila.In mammals, increasing evidence indicates that a HSD can

decrease longevity by inducing age-related chronic diseases such ashypertension, diabetes and heart failure (Stergiopoulos et al., 2009;Wei et al., 2018). In flies, it has also been shown that a strict HSD(fruit flies only ate a high-salt diet and drinking water was notprovided) can acutely accelerate mortality (Stergiopoulos et al.,2009). However, as we know, both animals and humans willincrease their water intake if they have eaten a salty diet. Therefore,it remains unknown whether a gentle HSD (the HSD flies wereprovided with pure water to drink every day) can affect lifespan ofDrosophila. In this experiment, the results showed that a 2%-SDsignificantly reduced the lifespan of Drosophila (P<0.05), and a4%-SD and an 8%-SD acutely reduced lifespan of flies (P<0.01,P<0.001, respectively) (Fig. 1K). These results suggested that agentle HSD could also reduce longevity of w1118 flies.In addition, the results showed that the decline in climbing ability

appeared to occur well before changes in mortality (Fig. 1I,K). Theadaption of climbing capacity and lifespan to exercise could beinfluenced by a HSD in aging Drosophila (Tinkerhess et al., 2012).A HSD may first damage the nervous system and skeletal musclesystem, such as by increasing oxidative stress. This may contributeto age-related diseases of the nervous system and skeletal musclesystem, which result in the age-related climbing ability decline(Bernardi et al., 2012; Lenda and Boegehold, 2002; Li et al., 2018;Petersen and Greene, 2008). Then, with the increase of age-relatedclimbing ability decline and oxidative stress, once they exceed thetolerances of their body, the flies will die in large numbers.Therefore, the end of declines in climbing may be a signal of thebeginning of a mass death of fruit flies.It has been reported that a HSD can increase oxidative stress in

cells and reduce salt tolerance, which can damage cell function andcause many diseases, such as skeletal muscle degeneration and heartfailure (Frisbee et al., 2000; Zhou et al., 2015). Increasing evidenceindicates that a HSD also decreases the exercise capacity inmammals. For instance, a HSD may impair muscle performance byinhibiting angiogenesis, increasing oxidative stress and enhancinginsulin resistance (Lenda and Boegehold, 2002; Li et al., 2018; Liuet al., 2014; Petersen and Greene, 2008). Moreover, it has beenreported that a HSD is an important cause of death in both humansand animals (Stergiopoulos et al., 2009; Xu et al., 2018b).However, in this study, it remained unclear whether a HSD thatdecreased climbing ability and lifespan was related to oxidativestress in aging flies.As we know, in both human and animals it is possible that

changing the composition of food can cause a decrease or increasein food intake in the short term. However, once individuals havebeen eating foods with altered ingredients for a long time, andbecome accustomed to this type of food, changes that increase ordecrease food intake because of a food composition alteration may

disappear. Therefore, previous studies almost ignore the secondaryeffect of reduced or increased food intake on the experimentalresults after changing the composition of a food, such as a high-fatdiet, a high-sugar diet, and a HSD in flies (Birse et al., 2010; Diopet al., 2017; Dobson et al., 2017; Na et al., 2013; Stergiopouloset al., 2009). Moreover, we think the long-term different salt dietswill not significantly affect our results via the secondary effect ofreduced or increased food intake.

The adaption of climbing capacity and lifespan to exercisecould be influenced by a HSD in aging DrosophilaIn both animals and humans, a number of studies report that exercisetraining can enhance skeletal muscle function, delay age-relatedskeletal muscle function decline and contribute to longevity(Behrend et al., 2012; Ferrara et al., 2008; Ozturk et al., 2017;Roh et al., 2016; Stanley et al., 2019; Wen et al., 2018). In addition,exercise training is considered an effective way to prevent and treatdiet-related obesity, potentially caused by high-fat diets and/orhigh-sugar diets (Lowman et al., 2018; Touati et al., 2010; Zhang,2017). For older or weak individuals, exercise training needs to bedone with great care and caution since these people are also prone tosports sickness and sports injuries (Kammerlander et al., 2012).However, it remains unclear whether exercise can prevent damageto climbing capacity and survival ability induced by the salt contentof a HSD.

The results showed that at 1 week old, exercise significantlyincreased the CFT of the 0%-SD, 2%-SD and 4%-SD flies (P<0.05,P<0.01, P<0.05, respectively) (Fig. 2A–C), but the CFT of 8%-SDflies did not change significantly after exercise training (P>0.05)(Fig. 2D). Furthermore, the results showed that at 3 weeks old,exercise significantly increased the CFT of the 0%-SD, 2%-SD and4%-SD flies (P<0.05, P<0.01, P<0.05, respectively) (Fig. 2E–G),but the CFT of 8%-SD flies did not change significantly afterexercise training (P>0.05) (Fig. 2H). Moreover, the results showedthat at 5 weeks old, exercise significantly increased the CFT of the0%-SD and 2%-SD flies (P<0.05, P<0.05, respectively) (Fig. 2I,J),and the CFT of the 4%-SD flies and 8%-SD flies did not changesignificantly after exercise training (P>0.05) (Fig. 2K,L). Theresults showed that in 0%-SD and 1-week-old flies, 3-week-old fliesand 5-week-old flies, exercise training significantly increased theirCI (P<0.01, P<0.05, P<0.01, respectively), and senilitysignificantly decreased their CI (P<0.01) (Fig. 2M). In 2%-SDand 1-week-old flies, 3-week-old flies and 5-week-old flies,exercise training significantly increased their CI (P<0.05), andsenility significantly decreased their CI (P<0.01) (Fig. 2N). In 2%-SD and 1-week-old flies and 3-week-old flies, exercise trainingsignificantly increased their CI (P<0.05), but the CI of 4%-SD and5-week-old flies did not change significantly after exercise training(P>0.05), and senility significantly decreased their CI (P<0.01)(Fig. 2O). The CI of 8%-SD and 1-week-old flies, 3-week-old fliesand 5-week-old flies did not change significantly after exercisetraining (P>0.05), and senility significantly decreased their CI(P<0.01) (Fig. 2P). What is more, the results showed that in the 0%-SD flies and 2%-SD flies, exercise training significantly increasedtheir lifespan (P<0.05) (Fig. 2Q,U), but the lifespan of 4%-SD fliesand 8%-SD flies did not change significantly after exercise training(P>0.05) (Fig. 2V,W). These results suggested that aging and an8%-SD reduced the benefits of exercise training in regards toclimbing capacity and survival. Exercise training improved theHSD-induced impairment of climbing capacity and longevity inyoung and adult flies. However, the mechanism of these remainsunclear.

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In flies, oxidative stress is not only related to survival and aging,but also closely related to exercise (He and Jasper, 2014; Piccirilloet al., 2014). For instance, the amount of oxidative damage increasesas an organism ages, and it is postulated to be a major causal factor

of senescence. Overexpression of antioxidative enzymes retards theage-related accrual of oxidative damage and extends the maximumlifespan of transgenic D. melanogaster (Koh et al., 2012; Liu et al.,2018b; Mullinax et al., 2015). It has been reported that flight

Fig. 2. See next page for legend.

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training increases oxidative damage and reduces survival in flies.Climbing endurance training can enhance climbing ability bystrengthening skeletal muscles and mitochondrial activity in flies,which indicates that endurance training reduced the damage ofoxidative stress (Sujkowski and Wessells, 2018; Tinkerhess et al.,2012). In mammals, increasing evidence suggests that a HSDincreases the damage of oxidative stress and reduces antioxidantability in several ways (Lenda and Boegehold, 2002; Liu et al.,2014; Uetake et al., 2015). For example, high salt levels can increasereactive oxygen species (ROS) generation partly through theenzyme NADPH oxidase (Fellner et al., 2014; Liu et al., 2014).Furthermore, high salt levels can upset the balance of the lipidmetabolism, and this could produce a lot of malondialdehyde(MDA) (Catanozi et al., 2003). It has been reported that a HSD canincrease oxidative stress in cells and reduce salt tolerance, which candamage cell function and cause many diseases, such as skeletalmuscle degeneration and heart failure (Frisbee et al., 2000; Zhouet al., 2015). Exercise training can enhance the antioxidant ability ofskeletal muscle and cardiac muscle in humans and animals(Navarro-Arevalo et al., 1999; Rinaldi et al., 2006).In this study, the results showed that a 2%-SD, a 4%-SD and an

8%-SD significantly increased the expression of Salt gene (P<0.01),and the expression of Salt gene of 2%-SD flies, 4%-SD flies and8%-SD flies did not change significantly after exercise training(P>0.05) (Fig. 3A). In addition, the results showed that a 2%-SD, a4%-SD and an 8%-SD significantly decreased the expression ofdFOXO gene (P<0.01), and exercise training significantly increasedthe dFOXO gene expression in the 0%-SD flies, 2%-SD flies and4%-SD flies (P<0.01, P<0.01, P<0.05, respectively), but dFOXOgene expression of the 8%-SD flies did not change significantlyafter exercise training (P>0.05) (Fig. 3B). Moreover, the resultsshowed that a 2%-SD, a 4%-SD and an 8%-SD significantlydecreased the superoxide dismutase (SOD) level (P<0.01), andexercise training significantly increased the SOD level of the 0%-SD flies, 2%-SD flies and 4%-SD flies (P<0.01, P<0.01, P<0.05,respectively), but the SOD level of the 8%-SD flies did not changesignificantly after exercise training (P>0.05) (Fig. 3C). Finally, theresults showed that a 2%-SD, a 4%-SD and an 8%-SD significantlyincreased the MDA level (P<0.01, P<0.01, P<0.05, respectively),and exercise training significantly decreased the MDA level of the0%-SD flies, 2%-SD flies and 4%-SD flies (P<0.05), but the MDAlevel of the 8%-SD flies did not change significantly after exercisetraining (P>0.05) (Fig. 3D). These results suggest that exercise

training could improve the antioxidant ability in HSD young andadult flies, but this could not happen if the salt content of the dietwas 8%. Exercise training could not change the expression of Saltgene in Drosophila.

SOD and MDA are two common indexes for evaluating theability of eliminating oxygen free radicals (Yin et al., 2018). Alarge amount of SOD exists in the body, which eliminates freeradicals and has important roles in protecting the body fromdamage by ROS (Jing et al., 2015; Zhao et al., 2015). The SODcatalyzes the dismutation of superoxide into oxygen and hydrogenperoxide during physiological and pathological conditions,including aging (Cao et al., 2018). It has been demonstrated thatthe expression and activity of the SOD system are modified inaging, with reduced cell ability to counteract the oxidantmolecules, and consequently weak resistance to ROSaccumulation (Brown et al., 2005). Activation of FOXO isassociated with an increase in the expression of SOD and SODactivity (Klotz, 2017). MDA is commonly used as a marker of lipidperoxidation and is typically accessed via the thiobarbituric acidreactive substances assay, although this assay can be somewhatnonspecific as it can react with other aldehydes in addition toMDA. MDA is generated in vivo via peroxidation ofpolyunsaturated fatty acids (Duryee et al., 2010). The MDAtogether with excessive oxyradicals attack the cell membrane, andthus leads to cell necrosis (Chen et al., 2015; Cui et al., 2018; Liuet al., 2018a).

In both mammals and flies, it is well known that aging isaccompanied by a decline in locomotion ability (Demontis et al.,2013; Wen et al., 2016). In aging, oxidant production from somesources is increased, antioxidant enzymes are decreased and theadaptive response to oxidative stress is reduced (Liu and Marcinek,2017). Because of the increased oxidative stress levels in agedmuscles, ROS accumulation played an important role in musclechanges and sarcopenia (Liu and Marcinek, 2017; Navarro-Arevaloet al., 1999). Increasing evidence indicates that a HSD alsodecreases exercise capacity in mammals. For instance, a HSD mayimpair muscle performance by inhibiting angiogenesis, increasingoxidative stress and enhancing insulin resistance (Lenda andBoegehold, 2002; Li et al., 2018; Liu et al., 2014; Petersen andGreene, 2008). Moreover, it has been reported that a HSD is animportant cause of death in both humans and animals(Stergiopoulos et al., 2009; Xu et al., 2018b). Therefore, a HSDaccelerated the age-related decline of climbing capacity andmortality, and the mechanism of this may be that a HSD couldupregulate salt overexpression and decrease antioxidant capacity.

Recent studies report that endurance exercise protects flies fromobesity and lifespan reduction induced by a high-fat diet whenexercise and a high-fat diet are present at the same time (Wen et al.,2018). Endurance exercise can delay heart aging by enhancingantioxidant ability and NAD/SIR2 related pathway (Wen et al.,2019). Moreover, different CG9940 gene expression and PGC-1αexpression can affect the adaption of climbing ability and lifespan toexercise in flies (Tinkerhess et al., 2012; Wen et al., 2016).Therefore, in this study, this evidence indicates that exercise trainingcould improve the impairment of climbing capacity and longevityinduced by a HSD in young and adult flies, and the mechanism ofthis may be that exercise training enhances the antioxidant ability.However, exercise training could not effectively resist theimpairment of climbing capacity and longevity induced by an8%-SD and aging. We guess that too much salt in the diet and agingmay produce too much oxidative toxicity, which overloads thelimits to resilience of antioxidant capacity induced by exercise.

Fig. 2. The effect of exercise training on climbing capacity andmortality in HSD and aging flies. (A). Time to fatigue of 1-week-old andw1118 flies. (B) Time to fatigue of 1-week-old and 2%-SD flies. (C) Time tofatigue of 1-week-old and 4%-SD flies. (D) Time to fatigue of 1-week-old and8%-SD flies. (E) Time to fatigue of 3-week-old and w1118 flies. (F) Time tofatigue of 3-week-old and 2%-SD flies. (G) Time to fatigue of 3-week-old and4%-SD flies. (H) Time to fatigue of 3-week-old and 8%-SD flies. (I) Time tofatigue of 5-week-old and w1118 flies. (J) Time to fatigue of 5-week-old and2%-SD flies. (K) Time to fatigue of 5-week-old and 4%-SD flies. (L) Time tofatigue of 5-week-old and 8%-SD flies. (M) The climbing index in w1118flies. (N) The climbing index in 2%-SD flies. (O) The climbing index in 4%-SD flies. (P) The climbing index in 8%-SD flies. (Q) The curves of survivaland the average lifespan of w1118 flies. (R) The curves of survival and theaverage lifespan of 2%-salt-diet flies. (S) The curves of survival and theaverage lifespan of 4%-SD flies. (T) The curves of survival and the averagelifespan of 8%-SD flies. Using a non-parametric followed by a log-rank testfor analyze survival and time to fatigue. A two-way ANOVA was used toanalyze the effects of exercise and aging on climbing index of ‘no exercise’and ‘exercise’ flies. Data are represented as means±s.e.m. *P<0.05;**P<0.01.

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Endurance exercise improved the climbing capacity andsurvival in salt-overexpression DrosophilaIt has been reported that the salt gene is associated with salttolerance in flies. For example, a HSD reduces the salt tolerance andlifespan of flies, and it increases the expression of salt gene(Stergiopoulos et al., 2009). However, it remains unclear whetheroverexpression of the salt gene can affect the longevityand climbing capacity in flies, and whether exercise trainingcan improve the longevity and climbing capacity insalt-overexpression flies.In this study, the salt gene was overexpressed by UAS/arm-gal4

system. The results showed that in 1-week-old flies, overexpression ofthe salt gene significantly decreased the CFT (P<0.05), exercisetraining significantly increased the CFT (P<0.05) (Fig. 4A). In3-week-old flies, overexpression of salt gene also significantlydecreased the CFT (P<0.05), exercise training significantlyincreased the CFT (P<0.05) (Fig. 4B). However, in 5-week-oldflies, overexpression of the salt gene significantly decreased the CFT(P<0.01), exercise training could not be said to significantly increasethe CFT (P>0.05) (Fig. 4C). Next, in salt-control flies, salt-overexpression flies, and salt-overexpression+E flies, senilitysignificantly decreased their CI (P<0.01) (Fig. 4D). In 1-week-oldflies, overexpression of the salt gene significantly decreased the CI(P<0.05), but exercise training significantly increased the CI (P<0.05)(Fig. 4E). In 3-week-old flies, overexpression of the salt genesignificantly decreased the CI (P<0.01), but exercise trainingsignificantly increased the CI (P<0.01) (Fig. 4E). In 5-week-oldflies, overexpression of the salt gene also significantly decreased theCI (P<0.01), but exercise training significantly increased the CI

(P<0.01) (Fig. 4E). Moreover, in flies, overexpression of the salt genesignificantly decreased lifespan (P<0.01), but exercise trainingsignificantly increased lifespan (P<0.05) (Fig. 4F,G). These resultsindicated that exercise training could improve the climbing capacityand survival in the salt gene overexpression flies, but it remainsunclear whether the mechanism of this was related to oxidative stress.

Our results showed that in 5-week-old flies, UAS/arm-gal4system significantly increased the expression of the Salt gene(P<0.01), but exercise training did not significantly decrease theexpression of the Salt gene (P>0.05) (Fig. 4H). In addition, Saltgene overexpression significantly decreased the expression ofdFOXO gene (P<0.01), but exercise training significantly increasedthe expression of dFOXO gene (P<0.05) (Fig. 4I). Moreover, Saltgene overexpression significantly decreased the SOD level(P<0.01), but exercise training significantly increased the SODlevel (P<0.01) (Fig. 4J). Finally, the results showed that in 5-week-old flies, Salt gene overexpression significantly increased the MDAlevel (P<0.01), but exercise training significantly increased theMDA level (P<0.01) (Fig. 4K). These results indicated that Saltgene overexpression impaired the climbing capacity and survivalvia weakening antioxidant ability. However, exercise training couldimprove the climbing capacity and survival by enhancingantioxidant ability in salt gene overexpression flies.

The salt gene knockdown resisted the impairment ofclimbing ability and lifespan induced by an 8%-SDIt has been reported that the gut’s salt gene knockdown can improvesalt tolerance, and it can increase the survival of a HSD fly(Stergiopoulos et al., 2009). However, it remains unclear whether

Fig. 3. The effect of exercise training and HSD on the expression of Salt and antioxidant capacity in aged flies. (A) The salt expression. (B) ThedFOXO expression. (C) The SOD activity level. (D) The MDA level. Independent-sample t-tests were used to assess differences between the no exerciseand exercise flies. Data are represented as means±s.e.m. *P<0.05; **P<0.01.

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whole body salt gene knockdown can affect the longevity andclimbing capacity in flies.In this study, our results showed that in 1-week-old flies, 3-week-

old flies and 5-week-old flies, salt gene knockdown did not

significantly change the CFT (P>0.05), and a HSD also did notsignificantly reduce the CFT (P>0.05) (Fig. 5A–C). In salt-controlflies, salt-knockdown flies and salt-knockdown+HSD flies, senilitysignificantly decreased their CI (P<0.01) (Fig. 4D). However, in

Fig. 4. The effect of exercise training on climbing capacity and mortality in aging and salt-overexpression flies. (A) Time to fatigue of 1-week-old flies.(B) Time to fatigue of 3-week-old flies. (C) Time to fatigue of 5-week-old flies. (D) The climbing index changes with aging in salt-overexpression flies.(E) The climbing index. (F) The curves of survival. (G) The average lifespan. (H) The salt expression. (I) The dFOXO expression. (J) The SOD activity level.(K) The MDA level. Using a non-parametric followed by a log-rank test for analyze survival and time to fatigue. One-way ANOVA with LSD tests wereused to identify differences among the ‘salt-control’, ‘salt-overexpression’, and ‘salt-overexpression+exercise’ flies. Data are represented as means±s.e.m.*P<0.05; **P<0.01.

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1-week-old flies, 3-week-old flies and 5-week-old flies, salt geneknockdown did not significantly change the CI (P>0.05), and aHSD also did not significantly reduce the CI (P>0.05) (Fig. 5E).What is more, salt gene knockdown did not significantly change thelifespan (P>0.05), and a HSD also did not significantly reduce thelifespan (P>0.05) (Fig. 5F,G), which was similar to previous studies(Stergiopoulos et al., 2009). Next, the results displayed that in

5-week-old flies, salt gene RNAi significantly decreased the saltgene expression (P<0.01), but a HSD did not significantly increasedthe salt gene expression (P>0.05) (Fig. 4H). Finally, In 5-week-oldflies, salt gene knockdown did not significantly change the dFOXOgene expression level, SOD level and MDA level, and a HSD didnot significantly reduce the dFOXO gene expression level and SODlevel, and it did not significantly increase MDA level (P>0.05)

Fig. 5. The effect of HSD on climbing capacity and mortality in aging and salt-RNAi flies. (A) Time to fatigue of 1-week-old flies. (B) Time to fatigue of3-week-old flies. (C) Time to fatigue of 5-week-old flies. (D) The climbing index changes with aging in salt-overexpression flies. (E) The climbing index.(F) The curves of survival. (G) The average lifespan. (H) The salt expression. (I) The dFOXO expression. (J) The SOD activity level. (K) The MDA level.Using a nonparametric followed by a log-rank test for analyze survival and time to fatigue. One-way ANOVA with LSD tests were used to identify differencesamong the salt-control, salt-knockdown and salt-knockdown+HSD flies. Data are represented as means±s.e.m. *P<0.05; **P<0.01.

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(Fig. 4I–K). These results suggested that the salt gene knockdownresisted the impairment of climbing ability, lifespan and antioxidantability induced by a HSD.Two principal classes of manipulation are usually employed to

study gene function. Loss-of-function (LOF) approaches attempt toeliminate gene function partially or completely. Gain-of-function(GOF) approaches attempt to obtain functional information bycreating conditions where the gene is excessively or ectopicallyexpressed or its function exaggerated (Layalle et al., 2011;Robertson et al., 2002). In this study, we have identified that theSalt gene function takes part in regulating salt tolerance, anddownstream it regulates antioxidant function, climbing capacity andaging.

The dFOXO gene overexpression reduced the impairment ofclimbing ability and lifespan induced by 8%-SDIn animals, it has been identified that a HSD increases the oxidativestress and causes cell damage (Lenda and Boegehold, 2002; Liuet al., 2014). In flies, oxidative stress is a major factor that leads toaging and decline of skeletal muscle ability (Duan et al., 2016; Rushet al., 2007; Sun et al., 2013). SOD and MDA are two commonindexes for evaluating the ability of eliminating oxygen free radicals(Yin et al., 2018). Activation of FOXO protein is associated with anincrease in the expression of SOD and SOD activity (Klotz, 2017).Therefore, to further confirm whether exercise resistance to a HSD-induced impairment of exercise capacity and lifespan was related toantioxidant capacity, the dFOXO gene expression was also changedby UAS/Gal4 system in flies, and their CFT, CI and lifespan weremeasured.The results showed that in 1-week-old flies, dFOXO gene

overexpression significantly increased the CFT (P<0.05), and itsignificantly increased the CFT in HSD 1-week-old flies (P<0.001)(Fig. 6A). In 3-week-old flies, dFOXO gene overexpressionsignificantly increased the CFT (P<0.05), and it also significantlyincreased the CFT in HSD 3-week-old flies (P<0.001) (Fig. 6B). In5-week-old flies, dFOXO gene overexpression significantlyincreased the CFT (P<0.05), and it also significantly increasedthe CFT in HSD 5-week-old flies (P<0.01) (Fig. 6C). Moreover, indFOXO-control flies, dFOXO-control+HSD flies, dFOXO-overexpression flies, and salt-dFOXO+HSD flies, senilitysignificantly decreased their CI (P<0.01) (Fig. 4D). In 1-week-oldflies, overexpression of dFOXO gene significantly increased the CI(P<0.05), and it also significantly increased the CI in HSD 1-week-old flies (P<0.05) (Fig. 6E). In 3-week-old flies, overexpression ofdFOXO gene significantly increased the CI (P<0.05), and it alsosignificantly increased the CI in HSD 3-week-old flies (P<0.01)(Fig. 6E). In 5-week-old flies, overexpression of dFOXO genesignificantly increased the CI (P<0.01), and it also significantlyincreased the CI in HSD 5-week-old flies (P<0.01) (Fig. 6E).Furthermore, overexpression of dFOXO gene significantlyincreased the lifespan of flies (P<0.05), and it also significantlyincreased the lifespan of HSD flies (P<0.01) (Fig. 6F,G). Theseresults suggested that overexpression of dFOXO gene improved thesalt tolerance of climbing capacity and lifespan in HSD flies.However, it remains unclear whether overexpression of dFOXOgene enhances the salt tolerance by changing Salt gene expressionor antioxidant capacity.The results showed that in 5-week-old flies, UAS/arm-gal4

system significantly increased the expression of dFOXO gene(P<0.01), it also significantly increase the dFOXO gene expressionin HSD flies (P<0.01) (Fig. 6I). In addition, the results showed thatoverexpression of dFOXO gene did not significantly change the salt

gene expression (P>0.05), and it also significantly did not changethe salt gene expression in HSD flies (P>0.05) (Fig. 6H). However,overexpression of dFOXO gene significantly increased the SODlevel (P<0.01), and it also significantly increased the SOD level inHSD flies (P<0.01) (Fig. 6J). Finally, the results showedoverexpression of dFOXO gene significantly decreased the MDAlevel (P<0.01), and it also significantly increased the MDA level inHSD flies (P<0.01) (Fig. 6K). These results suggested thatoverexpression of dFOXO gene improved the salt tolerance,climbing capacity and lifespan in HSD flies by enhancingantioxidant capacity, rather than by decreasing salt gene expression.

Our results also showed that the dFOXO gene knockdownsignificantly decreased the CFT and CI in flies (P<0.05, or P<0.01)(Fig. 7A–E), and a HSD aggravated the CFT and CI reduction indFOXO gene knockdown flies (P<0.05, or P<0.01, or P<0.001)(Fig. 7A–E). Moreover, the dFOXO gene knockdown significantlyreduced survival (P<0.05), and a HSD aggravated the lifespanreduction in dFOXO gene knockdown flies (P<0.01) (Fig. 7F,G).However, dFOXO gene knockdown did not significantly reduce thesalt gene expression level (P>0.05), but a HSD significantlyincreased the salt gene expression in dFOXO gene knockdown flies(P<0.01) (Fig. 7H). Next, dFOXO gene RNAi significantlydecreased the dFOXO gene expression (P<0.01), and a HSDaggravated dFOXO gene expression reduction in dFOXO geneknockdown flies (P<0.01) (Fig. 4I). Similarly, dFOXO geneknockdown significantly decreased the SOD activity level(P<0.01), and a HSD aggravated SOD activity level reduction indFOXO gene knockdown flies (P<0.01) (Fig. 4J). Finally, dFOXOgene knockdown significantly increased the MDA level (P<0.01),and a HSD aggravated MDA level increase in dFOXO geneknockdown flies (P<0.01) (Fig. 4K). Therefore, these resultsindicated that the dFOXO gene knockdown decreased the salttolerance of climbing capacity and lifespan by reducing antioxidantcapacity, rather than by increasing salt gene expression. A HSDaggravated the reduction of salt tolerance of climbing capacity andlifespan via reducing antioxidant capacity and increasing salt geneexpression.

Increasing evidence indicates that the dFOXO gene function isclosely to lifespan in flies. For instance, the dFOXO overexpressiondecreased mortality and increased lifespan of flies via reducingoxidative stress, and the dFOXO is required both for transcriptionalchanges that mark the fly’s dietary history and for nutritionalprogramming of lifespan by excess dietary sugar (Blice-Baum et al.,2015; Dobson et al., 2017; Lee et al., 2017; Liu et al., 2012). Inaddition, endurance exercise can delay heart aging by activatingdFOXO/SOD pathway (Wen et al., 2019). In here, we identified thatthe dFOXO gene overexpression played an important role infighting the effects of a HSD on reduced exercise capacity and lifeexpectancy.

In conclusion, current results indicated that a HSD accelerated theage-related decline of climbing capacity and mortality viaupregulating salt overexpression and decreasing dFOXO/SODpathway. Increased dFOXO/SOD pathway activity played a keyrole in mediating endurance exercise resistance to low salt tolerance-induced impairment of climbing capacity and longevity in agingDrosophila.

MATERIALS AND METHODSFly stocks, diet and husbandryThe w1118 was a gift from Xiu-Shan Wu (Heart Development Center ofHunan Normal University, stock ID: 6326). The UAS-salt-overexpressionflies [stock ID: 58888. Genotype: w*; TI{TI}mir-1014KO saltmir-1014-KO],

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the UAS-dFOXO-overexpression flies (stock ID: 9575.Genotype: y1 w*;P{UAS-foxo.P}2), and the arm-gal4 (stock ID: 39249. Genotype: w1118;P{GMR60D12-GAL4}attP2) flies were obtained from the BloomingtonStock Center. The UAS-salt-knockdown flies (stock ID: v28349. Genotype:w1118; P{GD12732}v28349/TM3) and the UAS-dFOXO-knockdown flieswere obtained from the Vienna Drosophila Resource Center (stock ID:v30557. Genotype:w1118; P{GD4348}v30557).

To avoid the influence of genetic background differences on the results,maternal origin was used as the genetic control. The virgin female ‘w*; TI(Chang et al., 2019) mir-1014KO saltmir-1014-KO’ and ‘arm-gal4>w*; TI(Chang et al., 2019) mir-1014KO saltmir-1014-KO’ were represented as ‘salt-control’ and ‘salt-overexpression’. The female ‘y1 w*; P{UAS-foxo.P}2’and ‘arm-gal4>y1 w*;P{UAS-foxo.P}2’ were represented as ‘dFOXO-control’ and ‘dFOXO-overexpression’. The female ‘w1118;

Fig. 6. The effect of dFOXO overexpression on salt tolerance of HSD flies. (A) Time to fatigue of 1-week-old flies. (B) Time to fatigue of 3-week-old flies.(C) Time to fatigue of 5-week-old flies. (D) The climbing index changes with aging in salt-overexpression flies. (E) The climbing index. (F) The curves ofsurvival. (G) The average lifespan. (H) The salt expression. (I) The dFOXO expression. (J) The SOD activity level. (K) The MDA level. Using a non-parametric followed by a log-rank test for analyze survival and time to fatigue. Two-way ANOVA was used to identify differences among the four groups. Dataare represented as means±s.e.m. *P<0.05; **P<0.01; **P<0.001.

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P{GD12732}v28349/TM3’ and ‘arm-gal4>w1118; P{GD12732}v28349/TM3’ were represented as ‘salt-control’ and ‘salt-knockdown’.The female ‘w1118; P{GD4348}v30557’ and ‘arm-gal4>w1118;P{GD4348}v30557’ were represented as ‘dFOXO-control’ and‘dFOXO-knockdown’.

Normal food contained 10% yeast, 10% sucrose and 2% agar (Birse et al.,2010). To make a HSD, 2%, 4% and 8% of salt (NaCl) was added to normaldiets. When flies were fed a HSD, 1 ml of pure water was added to the

sponge stopper to drink every day (Fig. 1A). This is a gentle HSD comparedwith Stergiopoulos’ research since the HSD flies in their study had no waterto drink (Stergiopoulos et al., 2009). In addition, for normal fly diets, wealso added 1 ml of pure water to the sponge stopper every day as control.During the experimental time course, all experimental virgin female flieswere housed in a 25°C incubator with 50% humidity and a 12-h light/darkcycle. Fresh food was provided every other day for the duration of theexperiment.

Fig. 7. The effect of dFOXO knockdown on salt tolerance of HSD flies. (A) Time to fatigue of 1-week-old flies. (B) Time to fatigue of 3-week-old flies. (C)Time to fatigue of 5-week-old flies. (D) The climbing index changes with aging in salt-overexpression flies. (E) The climbing index. (F) The curves of survival.(G) The average lifespan. (H) The salt expression. (I) The dFOXO expression. (J) The SOD activity level. (K) The MDA level. Using a non-parametricfollowed by a log-rank test for analyze survival and time to fatigue. One-way ANOVA with LSD tests were used to identify differences among the ‘dFOXO-control’, ‘dFOXO-knockdown’, and ‘dFOXO-knockdown+HSD’ flies. Data are represented as means±s.e.m. *P<0.05; **P<0.01.

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Exercise training device and protocolsWhen constructing the exercise device, the advantage of the flies’natural negative geotaxis behavior was used to induce upward walking(Tinkerhess et al., 2012). All exercise group flies started exercise fromwhen they were 2-days old, and underwent a 5-week-long exerciseprogram. When flies climbed and reached the top of a vial, the vialwould be inverted. For young and adult flies, vials were verticallyloaded in an exercise device, and rotated 180° to make flies constantlyclimb (Fig. 8A) ( just as Power Tower, overcoming weight=total bodyweight) (Tinkerhess et al., 2012). For older flies, vials were loaded in anexercise device, and their long axis was set at an angle of 45° to thehorizontal plane (overcoming weight=total body weight×sin45°). Whenolder flies climbed and reached the top of vial, the vial was rotated 90°to make flies constantly climb (Fig. 8B) ( just as Tread Wheel andplaying teeterboard) (Lowman et al., 2018; Sujkowski and Wessells,2018). This avoided the effects of drop down and rotation on flies whentraining. Flies were exercised in vials with a 10-cm length. Vials wererotated at 0.16 rev/s. After vials completed an up-and-down turn, theywere held for 15 s to allow the flies time to climb. Flies were exercisedfor 1.5 h per day and training consisted of 5 days exercise and resting2 days per week.

RunspanClimbing endurance was measured using the fatigue assay describedpreviously (Tinkerhesset al., 2012). Eight vials of flies from each cohortwere subjected to the fatigue assay at three time points: once on day 7, onceon day 21 and on day 35. For each assessment, the flies were placed on theexercise machine and made to climb until they were fatigued, or no longerresponded to the negative geotaxis stimulus. Monitored at 30 min intervals,a vial of flies was visually determined to be ‘fatigued’ when five or fewerflies could climb higher than 1 cm after four consecutive drops. A minimumof eight vials containing 20 flies each was used for each fatigue assessmentwith each vial plotted as a single datum. Runspan graphs with fewer thaneight data points indicate that two or more vials were scored as fatigued atthe same time. Each experiment was performed in duplicate or triplicate, andrun spans were scored blind to the experimental process when possible. Thetime from the start of the assay to the time of fatigue was recorded for eachvial, and the data analyzed using log-rank analysis in GraphPad Prism(San Diego, CA, USA).

Negative geotaxis assayThe climbing apparatus consisted of an 18-cm-long vial with an innerdiameter of 2.8 cm, and flies were allowed to adapt to the vial for 10 minbefore assessing negative geotaxis. Sponges were placed in the ends of thetube to prevent escape while allowing air exchange. With a light box behindthe vials, the rack was tapped down five times and on the fifth, a timed digitalcamera snapped a picture after 8 s. The extent of climbing could be analyzedvisually or by imaging software. Five pictures of each group were taken andaveraged to arrive at a fixed score for each vial. The total score for all the fliesin a vial was tallied, and then divided by the number of flies in the vial togenerate the CI for that trial. Each vial was subjected to five trials, and thenthe indexes from the five trials were averaged (Tinkerhess et al., 2012).

Lifespan assaysDead flies were recorded daily. Lifespan was estimated for each fly as thenumber of days alive from the day of eclosion to the day of death. Mean andmedian lifespan and survival curves were used to characterize the lifespan.The average lifespan=(day 1×dead numbers of this day+day 2×deadnumbers of this day+…day n×dead numbers of this day)/total deadnumbers. Sample sizes were 200–210 flies per group (He and Jasper, 2014).

MDA assayMDA levels in flies were determined using the thiobarbituric acid (TBA)method. Ten fresh-frozen flies were converted to homogenates in ahomogenizer filled with 1 ml PBS (pH 7.2–7.4). The homogenates werecentrifuged at 4°C for 15 min with a speed of 2000 r/min. The supernatantwas mixed with the reagents supplied in an MDA Assay Kit (NanjingJiancheng Bioengineering Corporation, China, A003-2) and incubated at95°C for 40 min. After cooling at room temperature, the mixture wascentrifuged at 4000 g for 10 min. The absorbance of the supernatant wasmeasured at 530 nm. All operations were according to the manufacturer’sinstructions. The MDA concentrations were expressed as nmol/ml. Allassays were repeated three times.

Measurement of SOD activityThe total SOD activity in flies was determined using the hydroxylaminemethod. Ten fresh-frozen flies were converted to homogenates in ahomogenizer filled with 1 ml PBS (PH7.2–7.4). The homogenates werecentrifuged at 4°C for 15 min with a speed of 2000 r/min. The supernatant

Fig. 8. An image of a vials’ rotation on an exercise device. (A) For young and adult flies vials were vertically loaded in exercise device, and rotated 180°to make flies constantly climb ( just as Power Tower, overcoming weight = total body weight). (B) For aged flies vials were loaded in the exercise device, andtheir long axis is at an angle of 45° to the horizontal plane (overcoming weight = total body weight xsin45°). When aged flies climbed and reached the top ofvial, the vial were rotated 90° to make flies constantly climb.

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was mixed with the reagents supplied in a SOD Assay Kit (NanjingJiancheng Bioengineering Corporation, China, A001-3). The mixture wasincubated at room temperature for 10 min, and the absorbance of thecompound was then measured at 550 nm. All operations were according tothe manufacturer’s instructions. Each preparation was tested in triplicate.SOD activity was expressed as U/ml. All assays were repeated three times.

qRT-PCRAbout ten flies were homogenized in Trizol. First, 10 μg of the total RNAwas purified by organic solvent extraction from the Trizol (TRIzol,Invitrogen). The purified RNA was treated with DNase I (RNase-free,Roche) and used to produce oligo dT-primed cDNAs (SuperScript II RT,Invitrogen), which were then used as templates for quantitative real-timePCR. The rp49 gene was used as an internal reference for normalizing thequantity of total RNAs. Real-time PCR was performed with SYBR greenusing an ABI7300 Real time PCR Instrument (Applied Biosystems), withthree biological replicates. Expression of the various genes was determinedby the comparative CT method (ABI Prism 7700 Sequence DetectionSystem User Bulletin #2, Applied Biosystems). Primer sequences of saltwere as follows: F: 5′-TTAATCGCAGGCGCGTCAGTG-3′; R: 5′-GGACGAGACCACCGTGTTAATCAG-3′. Primer sequences of dFOXOwere as follows: F: 5′-AACAACAGCAGCATCAGCAG-3′; R: 5′-CTGAACCCGAGCATTCAGAT-3′. Primer sequences of Rp49 were asfollows: F:5′-CTAAGCTGTCGCACAAATGG-3′; R:5′-AACTTCTTGA-ATCCGGTGGG-3′.

Statistical analysesThe one-way ANOVA with LSD tests were used to identify differencesamong the relevant groups. A two-way ANOVA was used to analyze theeffects of a HSD and dFOXO overexpression on flies. Using a non-parametric followed by a log-rank test for analyzing survival and time tofatigue. Analyses were performed using the Statistical Package for the SocialSciences (SPSS) version 16.0 forWindows (SPSS Inc, Chicago, USA), withstatistical significance set at P<0.05. Data are represented as means±s.e.m.

AcknowledgementsThe authors thank Xiu-Shan Wu (the Center for Heart Development, College of LifeScience, Hunan Normal University) for supplying Drosophila of w1118. The authorsalso thank Lan Zheng (Key Laboratory of Physical Fitness and ExerciseRehabilitation of Hunan Province, Hunan Normal University) for supplyingDrosophila of arm-gal4.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsMethodology: D.-T.W., W.-Q.W., W.-Q.H., S.-X.C., S.S.Z.; Writing - original draft:D.-T.W.; Project administration: D.-T.W.; Funding acquisition: S.-X.C.

FundingThis work is supported by the National Key Research and Development Plan (no.2018YFC1604405), National Natural Science Foundation of China (31471590).

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